Novel compounds

ABSTRACT

Novel bacterial genes, microorganisms and processes for improving the manufacture of 5R clavams, eg. clavulanic acid.

[0001] The present invention relates to novel bacterial genes and processes for improving the manufacture of clavams e.g. clavulanic acid. The present invention also provides novel organisms capable of producing increased amounts of clavulanic acid.

[0002] Microorganisms, in particular Streptomyces sp. produce a number of antibiotics including clavulanic acid and other clavams, cephalosporins, polyketides, cephamycins, tunicamycin, holomycin and penicillins. There is considerable interest in being able to manipulate the absolute and relative amounts of these antibiotics produced by the microorganism and accordingly there have been a large number of studies investigating the metabolic and genetic mechanisms of the biosynthetic pathways [Domain, A. L. (1990) “Biosynthesis and regulation of beta-lactam antibiotics.” In: 50 years of Penicillin applications, history and trends]. Many of the enzymes which carry out the various steps in the metabolic pathways and the genes which code for these enzymes are known.

[0003] Clavams can be arbitrarily divided into two groups dependent on their ring stereochemistry (5S and 5R clavams). The biochemical pathways for the biosynthesis of 5R and 5S clavams have not yet been fully elucidated but it has been suggested that they are derived from the same starter units (an as yet unidentified 3 carbon compound [Townsend, C. A. and Ho, M. F. (1985) J. Am. Chem. Soc. 107 (4), 1066-1068 and Elson, S. W. and Oliver, R. S. (1978) J. Antibiotics XXXI No.6, 568] and arginine [Valentine, B. P. et al (1993) J. Am Chem. Soc. 15, 1210-1211] and share some common intermediates [Iwata-Reuyl, D. and C. A. Townsend (1992) J. Am. Chem. Soc. 114: 2762-63, and Janc, J. W. et al (1993) Bioorg. Med. Chem. Lett. 3:2313-16].

[0004] Examples of 5S clavams include clavam-2-carboxylate (C2C), 2-hydroxymethytclavam (2HMC), 2-(3-alanyl)clavam, valclavam and clavaminic acid [GB 1585661, Rohl. F. et al. Arch. Microbiol. 147:315-320, U.S. Pat. No. 4,202,819] There are, however, few examples of 5R clavams and by far the most well known is the beta lactamase inhibitor clavulanic acid which is produced by the fermentation of Strepromyces clavuligerus. Clavulanic acid, in the form of potassium clavulanate is combined with the beta-lactam amoxycillin in the antibiotic AUGMENTIN (Trade Mark SmithKline Beecham). Because of this commercial interest, investigations into the understanding of clavam biosynthesis have concentrated on the biosynthesis of the 5R clavam, clavulanic acid, by S. clavuligerus. A number of enzymes and their genes associated with the biosynthesis of clavulanic acid have been identified and published. Examples of such publications include Hodgson, J. E. et al. Gene 166, 49-55 (1995), Aidoo, K. A. et al., Gene 147, 41-46 (1994), Paradkar, A. S. et al., J. Bact. 177(5), 1307-14 (1995). In contrast nothing is known about the biosynthesis and genetics of 5S clavams other than clavaminic acid which is a clavulanic acid precursor produced by the action of clavaminic acid synthase in the clavulanic acid biosynthetic pathway in S. clavuligerus.

[0005] Gene cloning experiments have identified that S. clavuligerus contains two clavaminic acid synthase isoenzymes, cas1 and cas2 [Marsh, E. N. et al Biochemistry 31, 12648-657, (1992)] both of which can contribute to clavulanic acid production under certain nutritional conditions [Paradkar, A. S. et al., J. Bact. 177(5), 1307-14 (1995)]. Clavaminic acid synthase activity has also been detected in other clavulanic acid producing micro-organsims, ie. S. jumonjinensis [Vidal, C. M., ES 550549, (1987)] and S. katsurahamanus [Kitano, K. et al., JP 53-104796, (1978)] as well as S. antibioticos, a producer of the 5S clavam, valclavam [Baldwin, J. E. et al., Tetrahedron Letts. 35(17), 2783-86, (1994)]. The latter paper also reported S. antibioticos to have proclavaminic acid amidino hydrolase activity, another enzyme known to be involved in clavulanic acid biosynthesis. All other genes identified in S. clavuligerus as involved in clavam biosynthesis have been reported to be required for clavulanic acid biosynthesis [Hodgson, J. E. et al., Gene 166, 49-55 (1995), Aidoo, K. A. et al., Gene 147, 41-46 (1994)] and as yet none have been reported which are specific for the biosynthesis of 5S clavams.

[0006] We have now identified certain genes which are specific for the biosynthesis of 5S clavams as exemplified by C2C and 2HMC in S. clavutligertis. Accordingly the present invention provides DNA comprising one or more genes which are specific for 5S clavam biosynthesis in S. clavuligerus and which are not essential for 5R clavam (e.g. clavulanic acid) biosynthesis.

[0007] By “gene” as used herein we also include any regulatory region required for gene function or expression. In a preferred aspect the DNA is as identified as FIG. 1. Preferably the DNA comprises the nucleotide sequences indicated in FIG. 1 designated as orfup3, orfup2, orfup1, orfdwn1, orfdwn2 and orfdwn3. The present invention also provides proteins coded by said DNA. The present invention also provides vectors comprising the DNA of the invention and hosts containing such vectors.

[0008] Surprisingly we have found that when at least one of the genes according to the invention is defective the amount of clavulanic acid produced by the organism is increased. Accordingly the present invention also provides processes for increasing the amount of clavulanic acid produced by a suitable microorganism. In one aspect of the invention the genes identified can be manipulated to produce an organism capable of producing increased amounts of clavam, suitably clavulanic acid. The findings of the present work also allow an improved process for the identification of organisms with higher clavulanic acid production comprising a preliminary screening for organisms with low or no 5S clavam production (for example by hplc and/or clavam bioassay as described in the examples herein).

[0009] Suitably the 5S clavam genes of the present invention can be obtained by conventional cloning methods (such as PCR) based on the sequences provided herein. The function of the gene can be interfered with or eliminated/deleted by genetic techniques such as gene disruption [Aidoo, K. A. et al., (1994), Gene, 147, 4146]., random mutagenesis, site directed mutagenesis and antisense RNA.

[0010] In a further aspect of the invention there are provided plasmids containing one or more defective genes, preferably the plasmids pCEC060, pCEC061, pCEC056 and pCEC057, described below.

[0011] Suitably, the plasmids of the invention are used to transform an organism such as S. clavuligerus, e.g. strain ATCC 27064 (which corresponds to S. clavuligerus NRRL 3585). Suitable transformation methods can be found in relevant sources including: Sambrook, J., Fritsch, E. F. and Mvaniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Hopwood, D. A. et al (1985), Genetic Manipulation of Streptomyces. A Cloning Manual, and Paradkar, A. S. and Jensen, S. E. (1995), J. Bacteriol. 177 (5): 1307-1314.

[0012] Strains of the species S. clavuligerus are used industrially to produce clavulanic acid (potassium clavulanate). Within the British and United States Pharmacopoeias for potassium clavulanate (British Pharmacopoeia 1993, Addendum 1994, p1362-3 and U.S. Pharmacopeia Official Monographs 1995, U.S. Pat. No. 23 NF18 p384-5) the amounts of the toxic 5S clavam, clavam-2-carboxylate, are specifically controlled.

[0013] Therefore in a further aspect of the invention there is provided an organism capable of producing high amounts of clavulanic acid but has been made unable to make C2C or capable of producing high amounts of clavulanic acid but able to make only low levels of C2C. Suitably the clavulanic acid producing organism contains one or more defective clavam genes, and is preferably the S. clavuligerus strain 56-1A, 56-3A, 57-2B, 57-1C, 60-1A, 60-2A, 60-3A, 61-1A, 61-2A, 61-3A, and 61-4A, described below. Such organisms are suitable for the production of clavulanic acid without the production of the 5S clavam, clavam-2-carboxylate or with significantly reduced production of clavam-2-carboxylate.

EXAMPLES

[0014] In the examples all methods are as in Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning A Laboratory Manual (2nd Edition), or Hopwood, D. A. et al. (1985) Genetic Manipulation of Streptomyces. A Cloning Manual, and Paradkar, A. S. and Jensen, S. E. (1995) J. Bacteriol. 177 (5): 1307-1314 unless otherwise stated.

[0015] I. DNA Sequencing of the Streptomyces clavuligerus Chromosome Upstream and Downstream of the Clavaminate Synthase Gene cas1.

[0016] A. Isolation of cas1.

[0017] To isolate chromosomal DNA fragments from Streptomyces clavuligerus NRRL 3585 encoding the gene for clavaminate synthase isozyme 1 (cas1) an oligonucleotide probe RMO1 was synthesised based on nucleotides 9-44 of the previously sequenced cas1 gene (Marsh, E. N., Chang, M. D. T. and Townsend, C. A. (1992) Biochemistry 31: 12648-12657). Oligonucleotides were constructed using standard methods on an Applied Biosystems 391 DNA Synthesiser. The sequence of RMO1, a 36-mer, was synthesised in the antiparallel sense to that published by Marsh et al (1992, ibid) RMO1 was radiolabelled with ³²P using standard techniques for end-labelling DNA oligonucleotides (Sambrook et al., 1989 ibid), and was used to screen a cosmid bank of Streptomyces clavuligerus genomic DNA by Southern hybridization as described by Stahl and Amann (In: Nucleic acid techniques in bacterial systematics. Ed. E. Stackebrandt and M. Goodfellow. Toronto: John Wiley and Sons, p. 205-248, 1991). The genomic bank of S. clavuligerus DNA, prepared in cosmid pLAFR3, was as described by Doran, J. L et al., (1990), J. Bacteriol. 172 (9), 4909-4918.

[0018] Colony blots of the S. clavuligerus cosmid bank were incubated overnight with radiolabelled RMO1 at 60° C. in a solution consisting of 5×SSC, 5× Denhardt's solution, and 0.5% SDS (1×SDS: 0.15 M NaCl+0.015 M Na₃citrate; 1× Denhardt's solution: 0.02% BSA, 0.02% Ficoll, and 0.02% PVP). The blots were then washed at 68° C. for 30 minutes in a solution of 0.5×SSC+0.1% SDS. One cosmid clone, 10D7, was isolated that hybridised strongly to RMO1 and gave hybridization signals upon digestion with restriction endonucleases SacI and EcoRI that were consistent with hybridization signals detected in similar experiments with digests of S. clavuligerus genomic DNA. B. DNA sequencing of the S. clavuligerus chromosome flanking cas1.

[0019] A partial restriction map of cosmid 10D7 was generated using restriction endonucleases SacI, NcoI, and KpnI. Southern hybridization experiments between RMO1 and various digests of 10D7 DNA indicated that cas1 was most likely located at one end of a 7-kb SacI-SacI DNA subfragment. This fragment consisted of the cas1 open reading frame and approximately 6 kb of upstream DNA. The 7-kb fragment was then subcloned from a SacI digest of 10D7 in the phagemid vector pBluescriptII SK+ (2.96 kb; Stratagene), thus generating the recombinant plasmid pCEC007.

[0020] To facilitate the process of sequencing the chromosome upstream of cas1, a 3-kb NcoI-NcoI subfragment of the 7-kb SacI-SacI fragment was subcloned in pUC120 (3.2 kb; Vieirra and Messing, Methods Enzymol. 153, 3-11, 1987)) in both orientations, generating the recombinant plasmids pCEC026 and pCEC027. The 3-kb subfragment consisted of the amino-terminal-encoding portion of cast and approximately 2.6 kb of upstream DNA.

[0021] Nested, overlapping deletions were created in both pCEC026 and pCEC027 using exonuclease III and S1 nuclease digestion (Sambrook et al., 1989 ibid) and the DNA sequence of the 3-kb NcoI-NcoI fragment was determined on both strands by the dideoxy chain termination method (Sanger, F., Nicklen, S. and Coulson, A. R. (1977), Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467) using a Taq dye-deoxy^(a) terminator kit and an Applied Biosystems 373A Sequencer.

[0022] To determine the DNA sequence of the chromosome immediately downstream of cas1 a 4.3-kb KpnI-EcoRI DNA fragment was subcloned from cosmid clone 10D7 in pBluescriptII SK+, generating pCEC018. From pCEC018 a 3.7-kb SacI-SacI subfragment was cloned in pSL1180 (3.422 kb, Pharmacia); one of the SacI termini of this fragment partially overlapped the TGA stop codon of cas1, the other was vector encoded. Both orientations of the 3.7-kb fragment were obtained during subcloning and the resulting recombinant plasmids were designated pCEC023 and pCEC024. Nested, overlapping deletions were created in both plasmids and the DNA sequence of the 3.7-kb fragment was determined on both strands. The nucleotide sequence of the S. clavuligerus chromosome generated in these experiments, including and flanking cas1 sequence is shown in FIG. 1.

[0023] II. Functional Analysis of the Open Reading Frames Flanking cas1.

[0024] Computer analysis of the DNA sequence upstream of cas1 predicted the presence of two complete orfs and one incomplete orf. All three orfs were located on the opposite DNA strand to cas1 and were thus oriented in the opposite direction. The first open reading frame, orfup1, was located 579 bp upstream of cas1 and encoded a polypeptide of 344 amino acids (aa). The second open reading frame, orfup2, was located at 437 bp beyond the 3′-end of orfup1 and encoded a 151 aa polypeptide. Beyond orfup2 is orfup3. The start codon of orfup3 overlaps the translational stop codon of orfup2, suggesting that the two orfs are translationally coupled. No translational stop codon for orfup3 was located on the 3-kb NcoI-NcoI fragment.

[0025] A similar analysis of the DNA sequence downstream of cas1 predicted the presence of two complete orfs and one incomplete orf. Two of the orfs were located on the opposite DNA strand to cas1 and were thus oriented towards cas1. The third orf was located on the same strand as cas1 and was thus oriented away from it. The first downstream open reading frame, orfdwn1, was located 373 bp downstream of cas1 and encoded a 328 aa polypeptide. The second open reading frame, orfdwn2, was located 55 bp upstream of orfdwn1 and encoded a 394 aa polypeptide. At 315 bp upstream of orfdwn2 and on the opposite strand was orfdwn3. Because no stop codon was observed for orfdwn3 on the 3.7-kb fragment, it encoded an incomplete polypeptide of 219 aa.

[0026] Gene Disruption of the orfup and orfdwn Open Reading Frames

[0027] To assess the possible roles of the open reading frames flanking cas1 in the biosynthesis of clavulanic acid and the other clavams produced by S. clavuligerus, insertional inactivation or deletion mutants were created by gene replacement. The method used for gene disruption and replacement was essentially as described by Paradkar and Jensen (1995 ibid).

[0028] A. orfup1

[0029] A 1.5-kb NcoI-NcoI fragment carrying the apramycin resistance gene (apr^(r)), constructed as described in Paradkar and Jensen (1995 ibid), was treated with Klenow fragment to generate blunted termini (Sambrook et al., 1989 ibid) and was ligated to pCEC026 that had been digested with BsaBI and likewise treated with Klenow fragment. pCEC026 possesses a BsaBI site located within orfup1 at 636 bp from the translational start codon. The ligation mixture was used to transform competent cells of E. coli GM 2163 (available from New England Biolabs, USA., Marinus, M. G. et al M G G (1983) vol 122, p288-9) to apramycin resistance. From the resulting transformants two clones containing plasmids pCEC054 and pCEC055 were isolated; by restriction analysis pCEC054 was found to possess the apr^(r)-fragment inserted in the same orientation as orfup1, while pCEC055 possessed it in the opposite orientation.

[0030] To introduce pCEC054 into S. clavuligerus, plasmid DNA was digested with BamHI and HindIII and ligated to the high-copy number Streptomyces vector pU486 (6.2 kb; Ward et al., (1986) Mol. Gen. Genet. 203: 468-478). The ligation mixture was then used to transform E. coli GM2163 competent cells to apramycin resistance. From the resulting transformants one clone, possessing the shuttle plasmid pCEC061, was isolated. This plasmid was then used to transform S. clavuligerus NRRL 3585. The resulting transformants were put through two successive rounds of sporulation on non-selective media and then replica plated to antibiotic containing media to identify apramycin-resistant and thiostrepton-sensitive transformants. From this process four putative mutants (61-1A, -2A, -3A and -4A) were chosen for further analysis.

[0031] To confirm that these putative mutants were disrupted in orfup1 genomic DNA was prepared from isolates 61-1A and 61-2A, digested with SacI and subjected to Southern blot analysis. The results of the Southern blot were consistent with a double cross-over having occurred and demonstrated that these mutants are true disruption replacement mutants in orfup1.

[0032] The mutants 61-1A, -2A, -3A and -4A were grown in Soya-Flour medium and their culture supernatants were assayed by HPLC for clavulanic acid and clavam production. The composition of the Soya-Flour medium and the method for assaying clavams by HPLC were as previously reported (Paradkar and Jensen, 1995 ibid) except that the running buffer for the HPLC assay consisted of 0.1 M NaH₂PO₄+6% methanol, pH 3.68 (adjusted with glacial acetic acid). The HPLC analysis indicated that none of the mutants produced detectable levels of clavam-2-carboxylate or 2-hydroxymethylclavam. Furthermore, when culture supernatants were bioassayed against Bacillus sp. ATCC 27860, using the method of Pruess and Kellett (1983, J. Antibiot. 36: 208-212)., none of the mutants produced detectable levels of alanylclavam. In contrast, HPLC assays of the culture supernatants showed that the mutants appeared to produce superior levels of clavulanic acid when compared to the wild-type (Table 1). TABLE 1 Clavulanic acid titre (CA) of orfup1 mutants in shake flask tests 70 HOURS 93 HOURS 70 HOURS CA ug/ 93 HOURS CA ug/ STRAIN CA ug/ml mg DNA CA ug/ml mg DNA NRRL 3585 #1  87  915 166 1963 NRRL 3585 #2  66  790 159 1842 61-1A 272 2894 439 6113 61-2A 199 2148 225 2928 61-3A  54  692 221 2585 61-4A  0   0 226 2422

[0033] B. orfdwn1 and orfdwn2

[0034] A deletion/replacement mutant in orfdwn1 and orfdwn2 was created by first digesting pCEC018 (7.3 kb) with NcoI and liberating a 1-kb subfragment containing most of orfdwn1 and a portion of orfdwn2. The digest was fractionated by agarose-gel electrophoresis and the 6.3-kb fragment was excised and eluted from the gel. This fragment was then ligated to an NcoI-NcoI DNA fragment carrying apr^(r) and used to transform E. coli XL1-Blue to apramycin resistance. One clone was obtained from this experiment but restriction analysis of the resulting recombinant plasmid revealed that two copies of the apramycin resistance fragment had been ligated into the deletion plasmid. To eliminate the extra copy of the apr^(r)-fragment, the plasmid was digested with NcoI and self-ligated. The ligation mixture was used to transform E. coli GM2163 to apramycin resistance. From the transformants, two clones were isolated that contained plasmids pCEC052 and pCEC053 both of which possessed only one copy of the apr^(r)-fragment; pCEC052 possessed the apr^(r)-fragment inversely oriented with respect to orfdwn1 and 2, while pCEC053 possessed the apr^(r)-fragment inserted in the same orientation as orfdwn1 and 2.

[0035] A shuttle plasmid of pCEC052 was constructed by ligating BamHI-digested pCEC052 with similarly digested pIJ486 and transforming E. coli GM2163 to apramycin resistance. From this experiment one clone was isolated that contained the shuttle plasmid pCEC060. This plasmid was used to transform wild-type S. clavuligerus 3585 to apramycin and thiostrepton resistance. The resulting transformants were put through two rounds of sporulation under non-selective conditions and then replica plated to antibiotic containing media to identify apramycin resistant, thiostrepton sensitive colonies. Three putative mutants (60-1A, -2A and -3A) were chosen for further analysis.

[0036] To establish the identity of these putative mutants genomic DNA was isolated from strains 60-1A and 60-2A and digested with either SacI or BstEII and subjected to southern blot analysis. The hybridisation bands generated from this experiment were consistent with both strains having undergone a double cross-over event demonstrating that these mutants are true disruption replacement mutants in orfdwn1/2.

[0037] When these were cultured in Soya-Flour medium and their culture supernatants assayed by HPLC, none of the mutants produced detectable levels of clavam-2-carboxylate or 2-hydroxymethylclavam. A bioassay of the culture supernatants showed that the mutants also failed to produce detectable levels of alanylclavam. As with the orfup1 mutants, the orfdwn1/2 mutants are capable of producing superior to wild-type levels of clavulanic acid (Table2). TABLE 2 Clavulanic acid titre (CA) of orfdwn1/2 mutants in shake flask tests 70 HOURS 93 HOURS 70 HOURS CA ug/ 93 HOURS CA ug/ STRAIN CA ug/ml mg DNA CA ug/ml mg DNA NRRL 3585 #1  87  915 166 1963 NRRL 3585 #2  66  790 159 1842 60-1A 164 1872 260 2911 60-2A 187 2013 108 1320 60-3A  79  994 214 2161

[0038] orfdwn3

[0039] To disrupt orfdwn3 pCEC023 (consisting of a 3.7-kb fragment of cas1 downstream DNA subcloned into pSL1180) was digested with NcoI and then self ligated. After transforming E. coli with the ligation mixture a clone was isolated that possessed the plasmid pCEC031. This plasmid retained only the 1.9 kb NcoI-EcoRI fragment encoding a portion of orfdwn2 and the incomplete orfdwn3. An examination of the DNA sequence revealed that pCEC031 possessed a unique BstEII site at 158 bp from the translational start site of orfdwn3. Therefore, pCEC031 was digested with BstEII, treated with Klenow fragment to create blunt ends and then ligated to a blunted apramycin resistance cassette. The ligation mixture was used to transform E. coli GM2163 to apramycin resistance and ampicillin resistance. Two transformants were selected that contained respectively pCEC050 and pCEC051. restriction analysis revealed that the apramycin resistance cassette was orientated in the same orientation as orfdwn3 in pCEC050 and in the opposite orientation in pCEC051. Both of these plasmids were then digested with HindIII and ligated to similarly digested pIJ486. The ligation mixtures were then used separately to transform E. coli GM2163 to apramycin and ampicillin resistance. The shuttle plasmids pCEC056 (pCEC050+pU486) and pCEC057 (pCEC051+pU486) were isolated from the resultant transformants. Both plasmids were then used to transform S. clavuligerus NR 3585.

[0040] One transformant was selected from each transformant experiment and put through two successive rounds of sporulation on non-selective media and then replica plated to antibiotic containing media to identify apramycin-resistant and thiostrepton-sensitive transformants. From this process two putative mutants were isolated from the progeny of each primary transformant. (56-1A and 56-3A for pCEC056, and 57-1C and 57-2B for pCEC057).

[0041] To establish the identity of these putative mutants genomic DNA was isolated from these strains and digested with either SacI or Acc65I and subjected to Southern blot analysis. The hybridisation bands generated from this experiment were consistent with both strains having undergone a double cross-over event demonstrating that these mutants are true disruption replacement mutants in orfdwn3.

[0042] When these strains were cultured in Soya-Flour medium and their culture supernatants assayed by HPLC, the mutants produced greatly reduced levels of clavam-2-carboxylate or 2-hydroxymethylclavam. A bioassay of the culture supernatants showed that the mutants also failed to produce detectable levels of alanylclavam. As with the orfup1 and orfdwn1/2 mutants, the orfdwn3 mutants were capable of producing superior to wild-type levels of clavulanic acid (Table 3). TABLE 3 Clavulanic acid titre (CA) of orfdwn3 mutants in shake flask tests 71 HOURS 93 HOURS 71 HOURS CA ug/ 93 HOURS CA ug/ STRAIN CA ug/ml mg DNA CA ug/ml mg DNA NRRL 3585 180 1580 193 1790 #1A NRRL 3585 179 1640 266 2310 #1B 56-1A  34  110 235 2160 56-3A 225 2140 274 2740 57-1C 253 2910 277 2920 57-2B 242 2240 193 1860

[0043] The application discloses the following nucleotide sequences:

[0044] SEQ ID No. 1: DNA sequence of FIG. 1

[0045] SEQ ID No. 2: orfup3 sequence

[0046] SEQ ID No. 3: orfup2 sequence

[0047] SEQ ID No. 4: orfup1 sequence

[0048] SEQ ID No. 5: orfdwn1 sequence

[0049] SEQ ID No. 6: ofrdwn2 sequence

[0050] SEQ ID No. 7: orfdwn3 sequence

1 19 1 7193 DNA Streptomyces clavuligerus 1 ccatggcggg cggcggctgc cccggagcct cggccggacc ggtgaccagg accaccccgg 60 tgggatagtg gcccgccacc cggcgcagca gactcccgga cacggacccg tgggtgtgcg 120 cggaaaggcc cggaggccgg gtcacagcca cgggtaacgc gcggtgtcct tgcccgcgta 180 atcggggtcc agatagacga aggcccggtg gacgaggaag tcccgcacct cgtagaccgt 240 gcaccagcgc ccggcggccc actcggggtc acccgcccgc cacggcccgt cccggtgctc 300 accgtgggtg gtgccctccg cggcgaggag ttcggtcccg gtcagaatcc agttgacgga 360 ccacagatgg tgggtgatcg agcggatggt gcccccgagg tcgtcgaaga gccgggcgat 420 ctcggacttg ccccgggcca gaccccactt ggggaagaag aagaccgcgt cctcggcgaa 480 gtagtcgatc gcgggggtgc cgtcgctgcc gacgccgccg ttgtcgaacg ccttgaagta 540 cgcggtgatg accgccttgc gctgctcgtc cgtcataccg gccgatgcca cggacatgaa 600 acgacctcca gagattccgg gtggctgtgc tggggctgcg gaaggggtgt cccccgcgaa 660 ggacggcgga cgccgcggac gccgcggccg tctccccggc ggacgggtcc cagcgtcctg 720 gagagggctt ggcggcggct tgacgccgtg ctgtcccgcg gcttgcggaa cgcgaagtac 780 cggccagcgt acgggcgttg caccggacgt gtacgccggt cgggacccct cgtacccccg 840 gagccggccg accccggcgg ctccgggggt acggacgcgc cggaccggcc cgagcgagcc 900 ggacgggtcg gacggtgcgc gtggttccgg tgtgtcggac agctcggacg gaccggacgg 960 tgcgcgtggt tccggtgtgt cggacagctc ggacgggtcg gacggtgcgc gtggttccgg 1020 cacgccggac gggtcagttg ccgatcatgg cgagcaatgc cggggtgtac cgctccccgg 1080 acaccgggtg ggagatcgcg gccgtcacct ccgcgaggga ccggtcgtcc agccggatcg 1140 aggcggcggc gagattgtcc gcgagatggg ccgggttcgc ggtgcccggg atcgggacga 1200 cgtcctcgcc ccggtggtgc agccaggcga gcgcgagctg tgccagggtc agccccagac 1260 cgtccgcgac cgggcgcagc cggtgcagca acgagcggtt gcgcgcgagg gccggagcgc 1320 tgaaccgggg ctggccccgg cggaagtcct cgtcccccag atcgtcggtg gtgcggatgg 1380 tgccggtgag aaaaccccgt cccagagggg cgtaagcgac gatcccgatc cccagctccc 1440 ggcagacggg caccacctcg tcctcgatcc cgcgcgacca caggctccac tcgctctgca 1500 ccgccgtcac cgggtgcacc gcgtccgccc ggcgcagcgt ggccgcggag ggctcggaga 1560 gaccgagcct gcggaccttg ccctcgcgca ccagctcggc caccgcaccc acggtctcct 1620 cgatcggcac cgccgggtcc gtccagtgct ggtagtacag gtcgatgcgg tcggtgccga 1680 gacgacgcag ggaccgttcg caggccgcgc ggacgtagga cggctcgccg cacaagccct 1740 gggaggcgcc gtcggacgag cgcaccatgc cgaacttggt ggcgatcagc acctcgtccc 1800 ggcggcccgc gaccgcccgt ccgagcagct cctcaccggc gccgagcccc tggacgtcgg 1860 cggtgtccag cagggtgacc ccggcgtcga cggcggcgcg gatggtggcc gtcgcccggg 1920 cgcggtccgg gcgtccgtag aagtcggtgg tcggcaggca gccgagcccc tgggcactga 1980 ccggaaggtc ccgcagggcg cggaccggcg gacgcggaac cgcggcggac acggaaccgg 2040 ccggggactc gggcggagag cgggacatac ggaacctcca caggcggagc cgggaacggg 2100 acgagggcga ggacgggacg gaacgaagga gaggacggga cggacagcac ggacgggacg 2160 gacggaacgg agtcgggaac cggggggggt gaccggaacc gggccgtcct tggccctccc 2220 ccgtcctccc cgccatccgc cgttctcccc cgttccctct cccgtcctcc agccaacacc 2280 gccgcccttt ccaagcgctt gacacggcac cgacagccgc cgccgggcgc ccgatgggga 2340 cccgtgcccg ccggtgagcg gcggtgagcg ccggtacggg accccacgcg ccgccgcccg 2400 ggcgcccgcc agggcccgcg cggccacccc ggcccgcccc ggccggagcg gcgatccggg 2460 ccgctcgctg caagaggaac atccacagcc gcacaaggag cgctccgcac agtgggcacc 2520 acgtccgccc cgtcccccac accgtggccg gtccccaccg gacagcacag caccgcacag 2580 caccacatcg cacggcacag cacagcacca ccggcacgag gaaccaagga aaggaaccac 2640 accaccatga cctcagtgga ctgcaccgcg tacggccccg agctgcgcgc gctcgccgcc 2700 cggctgcccc ggaccccccg ggccgacctg tacgccttcc tggacgccgc gcacacagcc 2760 gccgcctcgc tccccggcgc cctcgccacc gcgctggaca ccttcaacgc cgagggcagc 2820 gaggacggcc atctgctgct gcgcggcctc ccggtggagg ccgacgccga cctccccacc 2880 accccgagca gcaccccggc gcccgaggac cgctccctgc tgaccatgga ggccatgctc 2940 ggactggtgg gccgccggct cggtctgcac acggggtacc gggagctgcg ctcgggcacg 3000 gtctaccacg acgtgtaccc gtcgcccggc gcgcaccacc tgtcctcgga gacctccgag 3060 acgctgctgg agttccacac ggagatggcc taccaccggc tccagccgaa ctacgtcatg 3120 ctggcctgct cccgggccga ccacgagcgc acggcggcca cactcgtcgc ctcggtccgc 3180 aaggcgctgc ccctgctgga cgagaggacc cgggcccggc tcctcgaccg gaggatgccc 3240 tgctgcgtgg atgtggcctt ccgcggcggg gtggacgacc cgggcgccat cgcccaggtc 3300 aaaccgctct acggggacgc ggacgatccc ttcctcgggt acgaccgcga gctgctggcg 3360 ccggaggacc ccgcggacaa ggaggccgtc gccgccctgt ccaaggcgct cgacgaggtc 3420 acggaggcgg tgtatctgga gcccggcgat ctgctgatcg tcgacaactt ccgcaccacg 3480 cacgcgcgga cgccgttctc gccccgctgg gacgggaagg accgctggct gcaccgcgtc 3540 tacatccgca ccgaccgcaa tggacagctc tccggcggcg agcgcgcggg cgacgtcgtc 3600 gccttcacac cgcgcggctg agctcccggg tccgacaccg cgcggctgaa cccacggtcc 3660 ggggcccacg gtccggcacc gcgcggctga gcccccgggt ccggcagcgg gcggctgaac 3720 ccccgccccg ggccaccgcc cgaccgcccc cgcgcaccgg acgcgcccgc ctgtacggcg 3780 gtcccgcccg ggcccgtaca cctgaagcgc ccggcggacc gccgccccgc cgggggacgg 3840 acagagccgg gtgcgggagg acgtcctccc gcacccggct cccaccgttc cgcaccgacc 3900 gcacccgacc gtgccgcagg cgccaccggc accgcaccgc ccgcgccggc agccaccaca 3960 ggcgccacgc cgcccgcacg gtgcccgcgc tgctcagccc ccgtccaccg ggctgtccag 4020 cagccgccgc agcgcgcccc cgatgaactc ccggtcggcg gccgaccccc cggaccccgc 4080 gagatgcccc cacactcccg ggatcacctc cagcgaggca tacggcagca gatcggccac 4140 ccgcttctcg tcctcgacgg cgaaacacac gtccagggcg cccggcagca ccacggcccg 4200 cgccgtgacg gaggccagcg ccgcctcgac gctccccccg gccccgggtg tcgcccccac 4260 atccgtgttc tcccaggtgc gcaccatggt gagcagatcc gcggcgccgg gcccggagag 4320 gaagacctgc tcccagaagc cggtgaggta ctcctcgcgg gtggcgaaac ccagctcccg 4380 gtgggcacgg cgggcccaga aggaacgcga ggtcccccac ccggcgaaca cccggcccgc 4440 cgccttccgc ccccgctccc cggcgtcggc gctgagcgcc gcggccagac cggacagcag 4500 gaccaggctg tgcgggctgc tcaccggcgc cccgcagatc ggggcgatcc ggcgcaccat 4560 ccccggatgc gacacggccc actggtaggc gtgggccgcg cccatcgacc agcccgtgac 4620 cagggccagt tcccgtaccc ccagctcctc ggtgagcagc cggtgctgcg ccgcgacatt 4680 gtcctgcgga gtgatcagcg gaaagcggga ccccgacggg tggttgccgg gcgagctgga 4740 gaccccgttg ccgaagagtc cggcggtgac gacgcagtac cgccgggtgt ccagcggcag 4800 ccccgcaccg atcagccagt cgtacccggt gtggtcccgg ccgaagaacg acggacagag 4860 caccacgttc gtcccgtcgg cgttcggcgt gccgtacatg gcgtaaccga tccgggcgtc 4920 ccgcaggacc tccccgtcca gcaacggcag ttcgtcgatc tcgaatatgc ggcattccac 4980 cgctgacctc cttgttcgat ccccccggac aacaggtcgg tcgtggccgg agactcagag 5040 ccagttgggg gcgatctcgg tggcccacag ctccaggctg cgcagctgga catcgtgcgg 5100 gatcagcccg gagtactggc actggagcag atactccgga tcgtgccgct ccaccagctt 5160 ctcgatcatg cggttgatgt cgtccggggt gccgacccac tccagccccc ggtcgaccag 5220 ggtcttgtag tccgagccga tcggacccgt ctcgccggtc gcgcgcagcg cctcggtgaa 5280 gcccatgggg ccgaaccagt tctcgaagat gaagccgccg ccgcgggacg cccagtggtg 5340 ggcctcgccg gagtcccggg agaccaggac gtccttcatc accccgaccc gctcgccccg 5400 ccgcagggtg ccgtggcccg ccgcctcggc ctcctcccgg tagatgtcca tcagccgggc 5460 gacgatctgg tcgtcggtgt tcatcaggat cggcaccacg ccctcccggg cacagaaccg 5520 gaacgtgtcc tcactgaagc tgaacggctg gaagacgggc gggtgggggc gctggtaggg 5580 cttgggcgcg atgcccacct cgcggatgac gccgttctcg tcgaggcccc ggccgtagcg 5640 gcgcaccgcc tcgtagggga actccaggtc cggcaccggg atcgtccact gctccccgga 5700 gtgggtgaac gtctcggtcg tccacgcctt cttgatgatc tcccagtgct cctcgaagag 5760 ggcacgattg cgccggtccc gctccccggc gtcggacagg gtgccgccga ccccgtacac 5820 ctgccccatg atgtcggccc agcgcttctg gaacccgcgc gcgatcccga cgaaggcgcg 5880 gccccgggtc atgtggtcga gcatcgccag atcctcggcc agccgcagcg gattgtgcag 5940 cggcaggacg ttggccatct ggccgacccg gatgtgccgg gtctgcatgc cgaggtagag 6000 ccccagcatg atcgggttgt tggagacctc gaaaccctcg gtgtggaagt ggtgctcggt 6060 gaaggacagt ccccagtagc cgagttcgtc ggccgcctgc gcctgccggg tgagctgccg 6120 gagcatgttc tggtagttct gcggattgac ccccgccata ccccgctgga cctgcgcatg 6180 actgccgacc gttggcagat agaagagaat ggacttcacc ctggctcctc cggttcgcgg 6240 cgccctccat tgacgtgcgc cgaaagcggc tcgaccgtcc cactccgccc ttgagttccg 6300 tctgacgccg cgccagtcgg cgggccgtcc gccggggtgc ccgccggggt ccgcacccgc 6360 cggacggcac ggcgcgcacc gcgcgcgcgg cgcttcgggg caccgggctc gacggggtgc 6420 tcagcgggac gtccaacgga aggcaagccc ccgtacccag cctggtcaag gcgctcatcg 6480 ccattccctg aggaggtccc gccttgacca cagcaatctc cgcgctcccg accgtgcccg 6540 gctccggact cgaagcactg gaccgtgcca ccctcatcca ccccaccctc tccggaaaca 6600 ccgcggaacg gatcgtgctg acctcggggt ccggcagccg ggtccgcgac accgacggcc 6660 gggagtacct ggacgcgagc gccgtcctcg gggtgaccca ggtgggccac ggccgggccg 6720 agctggcccg ggtcgcggcc gagcagatgg cccggctgga gtacttccac acctggggga 6780 cgatcagcaa cgaccgggcg gtggagctgg cggcacggct ggtggggctg agcccggagc 6840 cgctgacccg cgtctacttc accagcggcg gggccgaggg caacgagatc gccctgcgga 6900 tggcccggct ctaccaccac cggcgcgggg agtccgcccg tacctggata ctctcccgcc 6960 ggtcggccta ccacggcgtc ggatacggca gcggcggcgt caccggcttc cccgcctacc 7020 accagggctt cggcccctcc ctcccggacg tcgacttcct gaccccgccg cagccctacc 7080 gccgggagct gttcgccggt tccgacgtca ccgacttctg cctcgccgaa ctgcgcgaga 7140 ccatcgaccg gatcggcccg gagcggatcg cggcgatgat cggcgagccg atc 7193 2 145 DNA Streptomyces clavuligerus 2 gtgacccggc ctccgggcct ttccgcgcac acccacgggt ccgtgtccgg gagtctgctg 60 cgccgggtgg cgggccacta tcccaccggg gtggtcctgg tcaccggtcc ggccgaggct 120 ccggggcagc cgccgcccgc catgg 145 3 453 DNA Streptomyces clavuligerus 3 atgtccgtgg catcggccgg tatgacggac gagcagcgca aggcggtcat caccgcgtac 60 ttcaaggcgt tcgacaacgg cggcgtcggc agcgacggca cccccgcgat cgactacttc 120 gccgaggacg cggtcttctt cttccccaag tggggtctgg cccggggcaa gtccgagatc 180 gcccggctct tcgacgacct cgggggcacc atccgctcga tcacccacca tctgtggtcc 240 gtcaactgga ttctgaccgg gaccgaactc ctcgccgcgg agggcaccac ccacggtgag 300 caccgggacg ggccgtggcg ggcgggtgac cccgagtggg ccgccgggcg ctggtgcacg 360 gtctacgagg tgcgggactt cctcgtccac cgggccttcg tctatctgga ccccgattac 420 gcgggcaagg acaccgcgcg ttacccgtgg ctg 453 4 1032 DNA Streptomyces clavuligerus 4 atgtcccgct ctccgcccga gtccccggcc ggttccgtgt ccgccgcggt tccgcgtccg 60 ccggtccgcg ccctgcggga ccttccggtc agtgcccagg ggctcggctg cctgccgacc 120 accgacttct acggacgccc ggaccgcgcc cgggcgacgg ccaccatccg cgccgccgtc 180 gacgccgggg tcaccctgct ggacaccgcc gacgtccagg ggctcggcgc cggtgaggag 240 ctgctcggac gggcggtcgc gggccgccgg gacgaggtgc tgatcgccac caagttcggc 300 atggtgcgct cgtccgacgg cgcctcccag ggcttgtgcg gcgagccgtc ctacgtccgc 360 gcggcctgcg aacggtccct gcgtcgtctc ggcaccgacc gcatcgacct gtactaccag 420 cactggacgg acccggcggt gccgatcgag gagaccgtgg gtgcggtggc cgagctggtg 480 cgcgagggca aggtccgcag gctcggtctc tccgagccct ccgcggccac gctgcgccgg 540 gcggacgcgg tgcacccggt gacggcggtg cagagcgagt ggagcctgtg gtcgcgcggg 600 atcgaggacg aggtggtgcc cgtctgccgg gagctgggga tcgggatcgt cgcttacgcc 660 cctctgggac ggggttttct caccggcacc atccgcacca ccgacgatct gggggacgag 720 gacttccgcc ggggccagcc ccggttcagc gctccggccc tcgcgcgcaa ccgctcgttg 780 ctgcaccggc tgcgcccggt cgcggacggt ctggggctga ccctggcaca gctcgcgctc 840 gcctggctgc accaccgggg cgaggacgtc gtcccgatcc cgggcaccgc gaacccggcc 900 catctcgcgg acaatctcgc cgccgcctcg atccggctgg acgaccggtc cctcgcggag 960 gtgacggccg cgatctccca cccggtgtcc ggggagcggt acaccccggc attgctcgcc 1020 atgatcggca ac 1032 5 984 DNA Streptomyces clavuligerus 5 gtggaatgcc gcatattcga gatcgacgaa ctgccgttgc tggacgggga ggtcctgcgg 60 gacgcccgga tcggttacgc catgtacggc acgccgaacg ccgacgggac gaacgtggtg 120 ctctgtccgt cgttcttcgg ccgggaccac accgggtacg actggctgat cggtgcgggg 180 ctgccgctgg acacccggcg gtactgcgtc gtcaccgccg gactcttcgg caacggggtc 240 tccagctcgc ccggcaacca cccgtcgggg tcccgctttc cgctgatcac tccgcaggac 300 aatgtcgcgg cgcagcaccg gctgctcacc gaggagctgg gggtacggga actggccctg 360 gtcacgggct ggtcgatggg cgcggcccac gcctaccagt gggccgtgtc gcatccgggg 420 atggtgcgcc ggatcgcccc gatctgcggg gcgccggtga gcagcccgca cagcctggtc 480 ctgctgtccg gtctggccgc ggcgctcagc gccgacgccg gggagcgggg gcggaaggcg 540 gcgggccggg tgttcgccgg gtgggggacc tcgcgttcct tctgggcccg ccgtgcccac 600 cgggagctgg gtttcgccac ccgcgaggag tacctcaccg gcttctggga gcaggtcttc 660 ctctccgggc ccggcgccgc ggatctgctc accatggtgc gcacctggga gaacacggat 720 gtgggggcga cacccggggc cggggggagc gtcgaggcgg cgctggcctc cgtcacggcg 780 cgggccgtgg tgctgccggg cgccctggac gtgtgtttcg ccgtcgagga cgagaagcgg 840 gtggccgatc tgctgccgta tgcctcgctg gaggtgatcc cgggagtgtg ggggcatctc 900 gcggggtccg gggggtcggc cgccgaccgg gagttcatcg ggggcgcgct gcggcggctg 960 ctggacagcc cggtggacgg gggc 984 6 1182 DNA Streptomyces clavuligerus 6 gtgaagtcca ttctcttcta tctgccaacg gtcggcagtc atgcgcaggt ccagcggggt 60 atggcggggg tcaatccgca gaactaccag aacatgctcc ggcagctcac ccggcaggcg 120 caggcggccg acgaactcgg ctactgggga ctgtccttca ccgagcacca cttccacacc 180 gagggtttcg aggtctccaa caacccgatc atgctggggc tctacctcgg catgcagacc 240 cggcacatcc gggtcggcca gatggccaac gtcctgccgc tgcacaatcc gctgcggctg 300 gccgaggatc tggcgatgct cgaccacatg acccggggcc gcgccttcgt cgggatcgcg 360 cgcgggttcc agaagcgctg ggccgacatc atggggcagg tgtacggggt cggcggcacc 420 ctgtccgacg ccggggagcg ggaccggcgc aatcgtgccc tcttcgagga gcactgggag 480 atcatcaaga aggcgtggac gaccgagacg ttcacccact ccggggagca gtggacgatc 540 ccggtgccgg acctggagtt cccctacgag gcggtgcgcc gctacggccg gggcctcgac 600 gagaacggcg tcatccgcga ggtgggcatc gcgcccaagc cctaccagcg cccccacccg 660 cccgtcttcc agccgttcag cttcagtgag gacacgttcc ggttctgtgc ccgggagggc 720 gtggtgccga tcctgatgaa caccgacgac cagatcgtcg cccggctgat ggacatctac 780 cgggaggagg ccgaggcggc gggccacggc accctgcggc ggggcgagcg ggtcggggtg 840 atgaaggacg tcctggtctc ccgggactcc ggcgaggccc accactgggc gtcccgcggc 900 ggcggcttca tcttcgagaa ctggttcggc cccatgggct tcaccgaggc gctgcgcgcg 960 accggcgaga cgggtccgat cggctcggac tacaagaccc tggtcgaccg ggggctggag 1020 tgggtcggca ccccggacga catcaaccgc atgatcgaga agctggtgga gcggcacgat 1080 ccggagtatc tgctccagtg ccagtactcc gggctgatcc cgcacgatgt ccagctgcgc 1140 agcctggagc tgtgggccac cgagatcgcc cccaactggc tc 1182 7 660 DNA Streptomyces clavuligerus 7 gtgcccggct ccggactcga agcactggac cgtgccaccc tcatccaccc caccctctcc 60 ggaaacaccg cggaacggat cgtgctgacc tcggggtccg gcagccgggt ccgcgacacc 120 gacggccggg agtacctgga cgcgagcgcc gtcctcgggg tgacccaggt gggccacggc 180 cgggccgagc tggcccgggt cgcggccgag cagatggccc ggctggagta cttccacacc 240 tgggggacga tcagcaacga ccgggcggtg gagctggcgg cacggctggt ggggctgagc 300 ccggagccgc tgacccgcgt ctacttcacc agcggcgggg ccgagggcaa cgagatcgcc 360 ctgcggatgg cccggctcta ccaccaccgg cgcggggagt ccgcccgtac ctggatactc 420 tcccgccggt cggcctacca cggcgtcgga tacggcagcg gcggcgtcac cggcttcccc 480 gcctaccacc agggcttcgg cccctccctc ccggacgtcg acttcctgac cccgccgcag 540 ccctaccgcc gggagctgtt cgccggttcc gacgtcaccg acttctgcct cgccgaactg 600 cgcgagacca tcgaccggat cggcccggag cggatcgcgg cgatgatcgg cgagccgatc 660 8 25 DNA Streptomyces clavuligerus 8 ctgacgctgc aggaggaagt cccgc 25 9 25 DNA Streptomyces clavuligerus 9 cggggcgagg acgtcgtccc gatcc 25 10 25 DNA Streptomyces clavuligerus 10 gagcccctgg acgtcggcgg tgtcc 25 11 25 DNA Streptomyces clavuligerus 11 gacggtgcat gctcagcagg gagcg 25 12 972 DNA Streptomyces clavuligerus 12 atgacctcag tggactgcac cgcgtacggc cccgagctgc gcgcgctcgc cgcccggctg 60 ccccggaccc cccgggccga cctgtacgcc ttcctggacg ccgcgcacac agccgccgcc 120 tcgctccccg gcgccctcgc caccgcgctg gacaccttca acgccgaggg cagcgaggac 180 ggccatctgc tgctgcgcgg cctcccggtg gaggccgacg ccgacctccc caccaccccg 240 agcagcaccc cggcgcccga ggaccgctcc ctgctgacca tggaggccat gctcggactg 300 gtgggccgcc ggctcggtct gcacacgggg taccgggagc tgcgctcggg cacggtctac 360 cacgacgtgt acccgtcgcc cggcgcgcac cacctgtcct cggagacctc cgagacgctg 420 ctggagttcc acacggagat ggcctaccac cggctccagc cgaactacgt catgctggcc 480 tgctcccggg ccgaccacga gcgcacggcg gccacactcg tcgcctcggt ccgcaaggcg 540 ctgcccctgc tggacgagag gacccgggcc cggctcctcg accggaggat gccctgctgc 600 gtggatgtgg ccttccgcgg cggggtggac gacccgggcg ccatcgccca ggtcaaaccg 660 ctctacgggg acgcggacga tcccttcctc gggtacgacc gcgagctgct ggcgccggag 720 gaccccgcgg acaaggaggc cgtcgccgcc ctgtccaagg cgctcgacga ggtcacggag 780 gcggtgtatc tggagcccgg cgatctgctg atcgtcgaca acttccgcac cacgcacgcg 840 cggacgccgt tctcgccccg ctgggacggg aaggaccgct ggctgcaccg cgtctacatc 900 cgcaccgacc gcaatggaca gctctccggc ggcgagcgcg cgggcgacgt cgtcgccttc 960 acaccgcgcg gc 972 13 48 PRT Streptomyces clavuligerus 13 Met Thr Arg Pro Pro Gly Leu Ser Ala His Thr His Gly Ser Val Ser 1 5 10 15 Gly Ser Leu Leu Arg Arg Val Ala Gly His Tyr Pro Thr Gly Val Val 20 25 30 Leu Val Thr Gly Pro Ala Glu Ala Pro Gly Gln Pro Pro Pro Ala Met 35 40 45 14 151 PRT Streptomyces clavuligerus 14 Met Ser Val Ala Ser Ala Gly Met Thr Asp Glu Gln Arg Lys Ala Val 1 5 10 15 Ile Thr Ala Tyr Phe Lys Ala Phe Asp Asn Gly Gly Val Gly Ser Asp 20 25 30 Gly Thr Pro Ala Ile Asp Tyr Phe Ala Glu Asp Ala Val Phe Phe Phe 35 40 45 Pro Lys Trp Gly Leu Ala Arg Gly Lys Ser Glu Ile Ala Arg Leu Phe 50 55 60 Asp Asp Leu Gly Gly Thr Ile Arg Ser Ile Thr His His Leu Trp Ser 65 70 75 80 Val Asn Trp Ile Leu Thr Gly Thr Glu Leu Leu Ala Ala Glu Gly Thr 85 90 95 Thr His Gly Glu His Arg Asp Gly Pro Trp Arg Ala Gly Asp Pro Glu 100 105 110 Trp Ala Ala Gly Arg Trp Cys Thr Val Tyr Glu Val Arg Asp Phe Leu 115 120 125 Val His Arg Ala Phe Val Tyr Leu Asp Pro Asp Tyr Ala Gly Lys Asp 130 135 140 Thr Ala Arg Tyr Pro Trp Leu 145 150 15 344 PRT Streptomyces clavuligerus 15 Met Ser Arg Ser Pro Pro Glu Ser Pro Ala Gly Ser Val Ser Ala Ala 1 5 10 15 Val Pro Arg Pro Pro Val Arg Ala Leu Arg Asp Leu Pro Val Ser Ala 20 25 30 Gln Gly Leu Gly Cys Leu Pro Thr Thr Asp Phe Tyr Gly Arg Pro Asp 35 40 45 Arg Ala Arg Ala Thr Ala Thr Ile Arg Ala Ala Val Asp Ala Gly Val 50 55 60 Thr Leu Leu Asp Thr Ala Asp Val Gln Gly Leu Gly Ala Gly Glu Glu 65 70 75 80 Leu Leu Gly Arg Ala Val Ala Gly Arg Arg Asp Glu Val Leu Ile Ala 85 90 95 Thr Lys Phe Gly Met Val Arg Ser Ser Asp Gly Ala Ser Gln Gly Leu 100 105 110 Cys Gly Glu Pro Ser Tyr Val Arg Ala Ala Cys Glu Arg Ser Leu Arg 115 120 125 Arg Leu Gly Thr Asp Arg Ile Asp Leu Tyr Tyr Gln His Trp Thr Asp 130 135 140 Pro Ala Val Pro Ile Glu Glu Thr Val Gly Ala Val Ala Glu Leu Val 145 150 155 160 Arg Glu Gly Lys Val Arg Arg Leu Gly Leu Ser Glu Pro Ser Ala Ala 165 170 175 Thr Leu Arg Arg Ala Asp Ala Val His Pro Val Thr Ala Val Gln Ser 180 185 190 Glu Trp Ser Leu Trp Ser Arg Gly Ile Glu Asp Glu Val Val Pro Val 195 200 205 Cys Arg Glu Leu Gly Ile Gly Ile Val Ala Tyr Ala Pro Leu Gly Arg 210 215 220 Gly Phe Leu Thr Gly Thr Ile Arg Thr Thr Asp Asp Leu Gly Asp Glu 225 230 235 240 Asp Phe Arg Arg Gly Gln Pro Arg Phe Ser Ala Pro Ala Leu Ala Arg 245 250 255 Asn Arg Ser Leu Leu His Arg Leu Arg Pro Val Ala Asp Gly Leu Gly 260 265 270 Leu Thr Leu Ala Gln Leu Ala Leu Ala Trp Leu His His Arg Gly Glu 275 280 285 Asp Val Val Pro Ile Pro Gly Thr Ala Asn Pro Ala His Leu Ala Asp 290 295 300 Asn Leu Ala Ala Ala Ser Ile Arg Leu Asp Asp Arg Ser Leu Ala Glu 305 310 315 320 Val Thr Ala Ala Ile Ser His Pro Val Ser Gly Glu Arg Tyr Thr Pro 325 330 335 Ala Leu Leu Ala Met Ile Gly Asn 340 16 328 PRT Streptomyces clavuligerus 16 Met Glu Cys Arg Ile Phe Glu Ile Asp Glu Leu Pro Leu Leu Asp Gly 1 5 10 15 Glu Val Leu Arg Asp Ala Arg Ile Gly Tyr Ala Met Tyr Gly Thr Pro 20 25 30 Asn Ala Asp Gly Thr Asn Val Val Leu Cys Pro Ser Phe Phe Gly Arg 35 40 45 Asp His Thr Gly Tyr Asp Trp Leu Ile Gly Ala Gly Leu Pro Leu Asp 50 55 60 Thr Arg Arg Tyr Cys Val Val Thr Ala Gly Leu Phe Gly Asn Gly Val 65 70 75 80 Ser Ser Ser Pro Gly Asn His Pro Ser Gly Ser Arg Phe Pro Leu Ile 85 90 95 Thr Pro Gln Asp Asn Val Ala Ala Gln His Arg Leu Leu Thr Glu Glu 100 105 110 Leu Gly Val Arg Glu Leu Ala Leu Val Thr Gly Trp Ser Met Gly Ala 115 120 125 Ala His Ala Tyr Gln Trp Ala Val Ser His Pro Gly Met Val Arg Arg 130 135 140 Ile Ala Pro Ile Cys Gly Ala Pro Val Ser Ser Pro His Ser Leu Val 145 150 155 160 Leu Leu Ser Gly Leu Ala Ala Ala Leu Ser Ala Asp Ala Gly Glu Arg 165 170 175 Gly Arg Lys Ala Ala Gly Arg Val Phe Ala Gly Trp Gly Thr Ser Arg 180 185 190 Ser Phe Trp Ala Arg Arg Ala His Arg Glu Leu Gly Phe Ala Thr Arg 195 200 205 Glu Glu Tyr Leu Thr Gly Phe Trp Glu Gln Val Phe Leu Ser Gly Pro 210 215 220 Gly Ala Ala Asp Leu Leu Thr Met Val Arg Thr Trp Glu Asn Thr Asp 225 230 235 240 Val Gly Ala Thr Pro Gly Ala Gly Gly Ser Val Glu Ala Ala Leu Ala 245 250 255 Ser Val Thr Ala Arg Ala Val Val Leu Pro Gly Ala Leu Asp Val Cys 260 265 270 Phe Ala Val Glu Asp Glu Lys Arg Val Ala Asp Leu Leu Pro Tyr Ala 275 280 285 Ser Leu Glu Val Ile Pro Gly Val Trp Gly His Leu Ala Gly Ser Gly 290 295 300 Gly Ser Ala Ala Asp Arg Glu Phe Ile Gly Gly Ala Leu Arg Arg Leu 305 310 315 320 Leu Asp Ser Pro Val Asp Gly Gly 325 17 394 PRT Streptomyces clavuligerus 17 Met Lys Ser Ile Leu Phe Tyr Leu Pro Thr Val Gly Ser His Ala Gln 1 5 10 15 Val Gln Arg Gly Met Ala Gly Val Asn Pro Gln Asn Tyr Gln Asn Met 20 25 30 Leu Arg Gln Leu Thr Arg Gln Ala Gln Ala Ala Asp Glu Leu Gly Tyr 35 40 45 Trp Gly Leu Ser Phe Thr Glu His His Phe His Thr Glu Gly Phe Glu 50 55 60 Val Ser Asn Asn Pro Ile Met Leu Gly Leu Tyr Leu Gly Met Gln Thr 65 70 75 80 Arg His Ile Arg Val Gly Gln Met Ala Asn Val Leu Pro Leu His Asn 85 90 95 Pro Leu Arg Leu Ala Glu Asp Leu Ala Met Leu Asp His Met Thr Arg 100 105 110 Gly Arg Ala Phe Val Gly Ile Ala Arg Gly Phe Gln Lys Arg Trp Ala 115 120 125 Asp Ile Met Gly Gln Val Tyr Gly Val Gly Gly Thr Leu Ser Asp Ala 130 135 140 Gly Glu Arg Asp Arg Arg Asn Arg Ala Leu Phe Glu Glu His Trp Glu 145 150 155 160 Ile Ile Lys Lys Ala Trp Thr Thr Glu Thr Phe Thr His Ser Gly Glu 165 170 175 Gln Trp Thr Ile Pro Val Pro Asp Leu Glu Phe Pro Tyr Glu Ala Val 180 185 190 Arg Arg Tyr Gly Arg Gly Leu Asp Glu Asn Gly Val Ile Arg Glu Val 195 200 205 Gly Ile Ala Pro Lys Pro Tyr Gln Arg Pro His Pro Pro Val Phe Gln 210 215 220 Pro Phe Ser Phe Ser Glu Asp Thr Phe Arg Phe Cys Ala Arg Glu Gly 225 230 235 240 Val Val Pro Ile Leu Met Asn Thr Asp Asp Gln Ile Val Ala Arg Leu 245 250 255 Met Asp Ile Tyr Arg Glu Glu Ala Glu Ala Ala Gly His Gly Thr Leu 260 265 270 Arg Arg Gly Glu Arg Val Gly Val Met Lys Asp Val Leu Val Ser Arg 275 280 285 Asp Ser Gly Glu Ala His His Trp Ala Ser Arg Gly Gly Gly Phe Ile 290 295 300 Phe Glu Asn Trp Phe Gly Pro Met Gly Phe Thr Glu Ala Leu Arg Ala 305 310 315 320 Thr Gly Glu Thr Gly Pro Ile Gly Ser Asp Tyr Lys Thr Leu Val Asp 325 330 335 Arg Gly Leu Glu Trp Val Gly Thr Pro Asp Asp Ile Asn Arg Met Ile 340 345 350 Glu Lys Leu Val Glu Arg His Asp Pro Glu Tyr Leu Leu Gln Cys Gln 355 360 365 Tyr Ser Gly Leu Ile Pro His Asp Val Gln Leu Arg Ser Leu Glu Leu 370 375 380 Trp Ala Thr Glu Ile Ala Pro Asn Trp Leu 385 390 18 220 PRT Streptomyces clavuligerus 18 Met Pro Gly Ser Gly Leu Glu Ala Leu Asp Arg Ala Thr Leu Ile His 1 5 10 15 Pro Thr Leu Ser Gly Asn Thr Ala Glu Arg Ile Val Leu Thr Ser Gly 20 25 30 Ser Gly Ser Arg Val Arg Asp Thr Asp Gly Arg Glu Tyr Leu Asp Ala 35 40 45 Ser Ala Val Leu Gly Val Thr Gln Val Gly His Gly Arg Ala Glu Leu 50 55 60 Ala Arg Val Ala Ala Glu Gln Met Ala Arg Leu Glu Tyr Phe His Thr 65 70 75 80 Trp Gly Thr Ile Ser Asn Asp Arg Ala Val Glu Leu Ala Ala Arg Leu 85 90 95 Val Gly Leu Ser Pro Glu Pro Leu Thr Arg Val Tyr Phe Thr Ser Gly 100 105 110 Gly Ala Glu Gly Asn Glu Ile Ala Leu Arg Met Ala Arg Leu Tyr His 115 120 125 His Arg Arg Gly Glu Ser Ala Arg Thr Trp Ile Leu Ser Arg Arg Ser 130 135 140 Ala Tyr His Gly Val Gly Tyr Gly Ser Gly Gly Val Thr Gly Phe Pro 145 150 155 160 Ala Tyr His Gln Gly Phe Gly Pro Ser Leu Pro Asp Val Asp Phe Leu 165 170 175 Thr Pro Pro Gln Pro Tyr Arg Arg Glu Leu Phe Ala Gly Ser Asp Val 180 185 190 Thr Asp Phe Cys Leu Ala Glu Leu Arg Glu Thr Ile Asp Arg Ile Gly 195 200 205 Pro Glu Arg Ile Ala Ala Met Ile Gly Glu Pro Ile 210 215 220 19 324 PRT Streptomyces clavuligerus 19 Met Thr Ser Val Asp Cys Thr Ala Tyr Gly Pro Glu Leu Arg Ala Leu 1 5 10 15 Ala Ala Arg Leu Pro Arg Thr Pro Arg Ala Asp Leu Tyr Ala Phe Leu 20 25 30 Asp Ala Ala His Thr Ala Ala Ala Ser Leu Pro Gly Ala Leu Ala Thr 35 40 45 Ala Leu Asp Thr Phe Asn Ala Glu Gly Ser Glu Asp Gly His Leu Leu 50 55 60 Leu Arg Gly Leu Pro Val Glu Ala Asp Ala Asp Leu Pro Thr Thr Pro 65 70 75 80 Ser Ser Thr Pro Ala Pro Glu Asp Arg Ser Leu Leu Thr Met Glu Ala 85 90 95 Met Leu Gly Leu Val Gly Arg Arg Leu Gly Leu His Thr Gly Tyr Arg 100 105 110 Glu Leu Arg Ser Gly Thr Val Tyr His Asp Val Tyr Pro Ser Pro Gly 115 120 125 Ala His His Leu Ser Ser Glu Thr Ser Glu Thr Leu Leu Glu Phe His 130 135 140 Thr Glu Met Ala Tyr His Arg Leu Gln Pro Asn Tyr Val Met Leu Ala 145 150 155 160 Cys Ser Arg Ala Asp His Glu Arg Thr Ala Ala Thr Leu Val Ala Ser 165 170 175 Val Arg Lys Ala Leu Pro Leu Leu Asp Glu Arg Thr Arg Ala Arg Leu 180 185 190 Leu Asp Arg Arg Met Pro Cys Cys Val Asp Val Ala Phe Arg Gly Gly 195 200 205 Val Asp Asp Pro Gly Ala Ile Ala Gln Val Lys Pro Leu Tyr Gly Asp 210 215 220 Ala Asp Asp Pro Phe Leu Gly Tyr Asp Arg Glu Leu Leu Ala Pro Glu 225 230 235 240 Asp Pro Ala Asp Lys Glu Ala Val Ala Ala Leu Ser Lys Ala Leu Asp 245 250 255 Glu Val Thr Glu Ala Val Tyr Leu Glu Pro Gly Asp Leu Leu Ile Val 260 265 270 Asp Asn Phe Arg Thr Thr His Ala Arg Thr Pro Phe Ser Pro Arg Trp 275 280 285 Asp Gly Lys Asp Arg Trp Leu His Arg Val Tyr Ile Arg Thr Asp Arg 290 295 300 Asn Gly Gln Leu Ser Gly Gly Glu Arg Ala Gly Asp Val Val Ala Phe 305 310 315 320 Thr Pro Arg Gly 

1. DNA comprising one or more genes specific for 5S clavam biosynthesis in S. clavuligerus and which is not essential for 5R clavam biosynthesis.
 2. DNA according to claim 1 as identified in FIG. 1 (SEQ ID No: 1).
 3. DNA according to claim 1 having the sequence or substantially the sequence shown in FIG. 1 as orfup3, orfup2, orfup1, orfdwn1, orfdwn2 or orfdwn3 (SEQ ID Nos: 2-7).
 4. DNA according to claim 1 having the sequence or substantially the sequence shown in FIG. 1 as orfup1 (SEQ ID No: 4).
 5. DNA which hybridises under conditions of high stringency with the DNA of claim
 1. 6. A vector comprising the DNA of claim 1 in which one or more of the genes specific for 5S clavam biosynthesis has been disrupted or otherwise made defective.
 7. A vector according to claim 6 containing one or more defective genes which is pCEC060, pCEC061, pCEC056 or pCEC057.
 8. A vector according to claim 7 which is pCEC061.
 9. A host containing the vector of claim
 6. 10. A host according to claim 9 which is capable of producing raised levels of clavulanic acid.
 11. A host according to claim 9 which is capable of producing low or no levels of 5S clavam.
 12. A host according to claim 9 which is S. clavuligerus.
 13. S. clavuligerus comprising DNA corresponding to an open reading frame flanking cas1 which DNA has been disrupted or otherwise made defective.
 14. S. clavuligerus according to claim 13 wherein the open reading frame is selected from the group consisting of orfup3, orfup2, orfup1, orfdwn1, orfdwn2 and orfdwn3.
 15. A process for improving 5R clavam production in a suitable microorganism comprising manipulation of DNA as defined in claim 1 and its inclusion in the microorganism.
 16. A process according to claim 15 wherein said suitable microorganism is S. clavuligerus.
 17. A process for improving 5R clavam production in S. clavuligerus comprising disrupting or otherwise making defective DNA regions flanking cas1.
 18. A process according to claim 15 wherein said DNA corresponds to open reading frames selected from the group consisting of orfup3, orfup2, orfup1, orfdwn1, orfdwn2 and orfdwn3.
 19. A process according to claim 15 wherein said DNA corresponds to open reading frame orfup1.
 20. A process according to claim 15 wherein said 5R clavam is clavulanic acid.
 21. A process for the identification of a microorganism suitable for high 5R clavam production comprising a preliminary screening for microorganisms with low or no 5S clavam production.
 22. A process according to claim 21 wherein the microorganism is S. clavuligerus.
 23. A process according to claim 22 wherein the 5R clavam is clavulanic acid.
 24. A process according to claim 21 wherein one or more genes specific for the production of 5S clavams is defective.
 25. A microorganism which is capable of 5R clavam production and low or no 5S clavam production obtainable by the process of claim
 15. 26. A microorganism obtainable by the process of claim 25 which is capable of producing clavulanic acid but which does not produce clavam-2-carboxylate.
 27. A microorganism obtainable by the process of claim 25 which is capable of producing clavulanic acid but which does not produce 2-hydroxymethylclavam.
 28. A microorganism obtainable by the process of claim 25 which is capable of producing clavulanic acid but which does not produce clavam-2-carboxylate and 2-hydroxymethylclavam.
 29. A microorganism obtained by the process of claim 15 which is strain 56-1A, 56-3A, 57-2B, 57-1C, 60-1A, 60-2A, 60-3A, 61-1A, 61-2A, 61-3A or 61-4A.
 30. Clavulanic acid obtainable by the fermentation of a microrganism as defined in claim
 25. 31. Clavulanic acid according to claim 30 which is free of clavam-2-carboxylate.
 32. Clavulanic acid according to claim 30 in the form of its potassium salt.
 33. Clavulanic acid which is free of any 5S clavam.
 34. Clavulanic acid which is free of any clavam-2-carboxylate.
 35. A composition comprising potassium clavulanate according to claim 32 in combination with a beta-lactam antibiotic.
 36. A composition according to claim 35 in which the beta-lactam antiobiotic is amoxycillin.
 37. A process for the preparation of a composition comprising potassium clavulanate and amoxycillin which process comprises producing clavulanic acid from a microorganism according to claim 25 and thereafter converting it to the potassium salt and combining the potassium salt with amoxycillin. 